Microbial sensitivity testing device



United States Patent Office Patented June 23, 1964 MICROBIAL SENSITIVITY TESTING DEVICE Bohdan Barna, Rexdale, Ontario, and James Gordon Thompson, Brampton, Ontario, Canada, assignors to British Drug Houses (Canada) Ltd., Toronto, Ontario,

Canada, a corporation of Canada No Drawing. Filed May 3, 1961, Ser. No. 107,337

7 Claims. (Cl. 195-1035)- The present invention relates to a device useful in the testing of chemical agents and is more particularly concerned with a testing device in tablet form in which the chemical agent to be tested is in intimate association with the test device. The invention further relates to the methods of making and using such a testing device in the determination of the sensitivity of micro-organisms to chemical agents, such as antibiotics, sulfonamides and other chemotherapeutic materials.

Heretofore, in order to determine the effectiveness of a chemical agent, such as an antibiotic or other medicament, against pathogenic organisms, it has been necessary to isolate the causative organism and to contact such chemical agent with the isolated organism in a proper culture medium. Upon the introduction of a new antibiotic or other chemical agent to the market, the manufacturer frequently provides a list of the organisms against which the antibiotic is effective and those against which it has no effect. However, within each species of bacteria, a number of strains will be resistant even though the majority are sensitive, or the opposite effect may be observed.

To aid in the determination of the effectiveness of the antibiotic, paper discs or tablets impregnated with the antibiotic have been used. This technique requires that the disc be implanted in or on the surface of a suitable culture medium which has been inoculated with the test organism. The culture medium, e.g. agar, is usually maintained in a petri dish or other suitable container. Upon incubation, the antibiotic, or other chemical agent previously impregnated in the disc, diffuses out into the culture medium. If the bacteria growing in the medium are resistant to the antibiotic they will grow right up to the edge of the disc impregnated with that antibiotic but if the bacteria are sensitive or susceptible to the antibiotic, a clear zone around the disc containing the antibiotic will appear. Usually, the diameter of the Zone of inhibition around the disc or tablet represents the degree of bacteriostatic or bacteriocidal activity of the selected antibiotic in the disc against the bacteria. or other organism on the surface of the culture medium. With different strains of the same organism, the larger the zone of inhibition, the more susceptible is that strain to the given antibiotic, all other factors in the test being equal. It is usually necessary to use several individual discs, each impregnated with different antibiotics, in a given culture medium containing the isolated organism in order to determine which antibiotic or chemical agent is to be used in the treatment of the particular bacterial infection. More recently, a multiple disc, consisting of a series of interconnected discs, each impregnated with a different antibiotic, has been fabricated from filter paper (see US. Patent Reissue The chief faults of conventional paper sensitivity discs, both single and multi-tipped, are the lack of uniformity in the potency between discs within a single lot and the failure of discs, even of correct and uniform potency of antibiotic, to release rapidly and uniformly the selected antibiotic on the surface of the culture medium.

The single disc type test devices are usually punched out of a specially selected filter or blotting paper that has been previously impregnated with the selected chemical agent by dipping the paper in an aqueous or other solution of the chemical agent, then excess moisture or solvent is removed and the disc then dried. Considerable migration and settling of the chemical agent can occur on the fibers of the paper during the drying step, yielding and uneven deposit of the selected chemical agent. Further, all filter or blotting papers inherently contain compressed fibers which are not packed uniformly or evenly. There is a tendency for the chemical agent to be impregnated by capillary action into the denser portions of the compressed fibers. As a result, heavy concentration of the antibiotic or other solute occurs in the center of the disc, with reduced concentrations being present at the periphery. Variation can be attributed to another aspect of the manufacturing technique employed. Paper disc impregnation is basically a batch technique in that a number of sheets of the filter or blotting paper are impregnated, sequentially or simultaneously, in the preparation of a single lot of discs. Similarly for multi-tipped discs a frame or clamp of the tips is immersed in the impregnating solution. Each manufacturing lot or batch is actually made up of a number of segments, i.e., a number of clamps full of tips, and these, although they are similarly treated, cannot of course be identically impregnate'd. One need only consider then the natural variation that occurs from sheet to sheet of the filter or blotting paper and in the case of multi-tipped discs not only the variation from sheet to sheet of the paper from which the discs have been cut, but also the degree of compression of the tips in the frames or clamps that held them during immersion. In both instances there are variations that result from the variation in the periods of immersion. Thus, the variation in potency of the chemical agent between discs produced within a single lot can be very great. This is reflected in the Government Regulations in both the United States and in Canada which allow an in-batch variation of 67 to 150% and plus or minus 40%, respectively. The latter means, for example, that within the same batch of discs, each designed to contain 10 units of penicillin, some discs could contain as little as 6 units while others could contain as much as 14 units of penicillin.

Frequently, the paper disc or tablet does not permit ready or uniform release of the chemical agent when the disc is placed in contact with the inoculated culture medium. This occurs because the paper fibersmay not be readily wettable and in some instances because the concentration of antibiotic varies greatly in area to area within each disc. As a result, in some paper discs the chemical agents may be eluted from only a portion of the disc, thereby making the results extremely difficult to interpret,

The use of sensitivity tablets has been suggested previously in order to eliminate some of the disadvantages of the paper discs. However, the tablets containing conventional sugars, binding agents, lubricating agents and surface active agents leave much to be desired. The diffusion or elution of the selected chemical agent into the culture medium is often poor, occasionally necessitating the use of from 10 to times the usual concentration of antibiotic agent in order to produce the desired result, Further, the tablets tend to fall off the surface of the culture medium. In practice, the disc or tablet is implanted on the surface of the inoculated culture medium, and the container or petri dish is inverted and maintained in the inverted position during the incubation period, eg- 16-18 hours. If the tablet or disc falls away from the surface of the culture medium, the entire test is, of course, vitiated. It has also been observed that many of the components of the tablets interfere with either the activity of the chemical agent or with the growth of the organism whose sensitivity is being determined.

Therefore, it is an object of the present invention to provide a device for use in testing a chemical agent that exhibits uniformity of potency or concentration of the chemical agent and which permits a rapid but reproducible rate of release of said chemical agent during the testing procedure.

A further object of the present invention is to provide an integral composition of matter in thin tablet form that readily adheres to the surface of a culture medium containing the micro-organism whose sensitivity is being determined.

Still another object of the present invention [5 to provide a test device for the determination of the sensitivity of micro-organisms to a chemical agent in which the chemical agent is maintained in stable form.

Another object of the present invention is to provide a method for the preparation of a testing device in thin tablet form.

It has been found that a testing device in thin tablet form can be prepared, with the chemical agent to be tested being associated intimately within said device, and which combines the advantages of the known paper discs and sensitivity tablets, respectively, while at the same time eliminating the major disadvantages known to exist in each of these test devices. In its essential form, the device of the present invention comprises an integral composition of matter in which the chemical agent is in admixture with a carrier and a binder, the carrier being preferentially wettable by water in relation to the said binder. Preferably, the test device is in extremely thin sheet or disc form, thus physically resembling prior art filter paper discs. These characteristics are achieved through the use of preferred chemical materials related to the cellulose. At the same time, the novel test device is prepared by tabletting means, thereby permitting the potency of the chemical agent to be accurately controlled within each tablet, yet unlike known sensitivity tablets the base of the present test device is essentially insoluble and the carrier and binder components comprising the base are essentially microbiologically and chemically inert. The carrier component of the tablets is preferentially wettable by water in relation to the binder, thereby permitting rapid abso ption of moisture from the culture medium with the result that the antibiotic or other chemical agent is rapidly eluted or diffused into the culture medium. The novel test device adheres readily to the surface of the culture medium. Preferably, the carrier is a highly purified wood cellulose material. The binder is composed preferably of a chemically modified cellulose derivative and while not readily wettable by water, does control the overall degree of liquid absorptiveness of the test device.

The test device and compositions of the present inven tion can thus be characterized as tablet discs since they resemble the size and consistency of paper discs while at the same time being prepared by essentially a tabletting method that imparts the advantages resulting from carefully controlled tablet procedures into the final product.

The invention further includes novel methods for the preparation and using of the test device. Preferably, the test device is made by first pro-mixing the antibiotic or other chemical agent with a microbiologically and chemically inert carrier, then admixing the resulting mixture with a microbiologically and chemically inert binder. and finally compressing the resulting admixture into a relatively thin tablet disc. This preferred procedure permits the active chemical agent to be readily dispersed onto the surface of the culture medium while the sensitivity test is being carried out.

The test devices, chemical compositions, and processes of this invention thus have utility as agents useful in the determination of the sensitivity of micro-organisms to chemical agents. Such chemical agents as antibiotics, sulfonamides and other chemotherapeutic agents, as well as amino acids, vitamins. carbohydrates, and other materials can all be tested for their effect on micro-organisms and like materials contained in appropriate culture media.

till

The term, chemical agent, as used in this specification and claims includes all substances, e.g. chemical biochemical, biological, and the like, whose effect upon contact with micro-organisms, organisms, and the like, is to be determined. The term is further deemed to include both the singular and plural sense and, hence, to include individual as well as combinations and mixtures of two or more agents.

In general, the tablet discs of the present invention are prepared by dissolving the antibiotic or other chemical agent in a suitable solvent and admixing the resulting solution directly with the selected binder and carrier. The admixing continues until homogeneity is obtained and the admixture is then dried and compressed into the desired tablets. This general procedure is operative and is satisfactory in virtually all instances where the concentration of the chemical agent per tablet disc is relatively high, as with sulfonamides, mandelarnines and many other chemotherapeutic agents.

However, since the utmost accuracy is usually desired and since the distribution of the antibiotic must be extremely uniform, it has been found advantageous to pre-mix the selected chemical agent with the carrier. In such preferred procedure, the antibiotic is first dissolved in a suitable solvent and added to a weighed quantity of the carrier, eg. purified wood cellulose. When the carrier is completely wetted with the antibiotic solution, it is spread thinly on a drying tray and subsequently dried under vacuum, using heat if the antibiotic or other chemical agent is thermostable. The resulting dried, antibiotic impregnated carrier is then passed through a fine mesh screen, e.g. number 60 mesh size, and then admixed with a selected amount, preferably an equivalent weight, of a dried. screened hinder, e.g. ethylccllulose. The resulting admixture is mixed thoroughly, assayed for potency, and then compressed into tablets, e.g. inch diameter tablets weighing 25 mg. each, having a hardness of about 5 kilograms. Preferably, the tablet discs are 0.75 to 1.25 millimeters in thickness, vs to /54 inch in diameter and weigh 25 milligrams or more, although many tablet discs exhibiting measurements outside these ranges are completely satisfactory. The resulting tablet discs are then dried under vacuum and stored under refrigeration in airtight containers, utilizing a suitable desiccant, so that the moisture content of the finished tablet discs remains below one percent (1% The following examples illustrate the preparation of the test. devices of the present invention but are not to be construed as limiting.

Example l.--r"cnicillin Tablet Disc (I 0 Units Each) The following components were utilized:

Parts by Penicillin G. potassium (in anhydrous reagent grade methanol containing 1120 units (40% overage) of penicillin per milliliter) "milliliters" 50.0

The penicillin was dissolved in the anhydrous methanol using clean volumetric glassware. The penicillin solution. in a ratio of one milliliter per gram of the purified wood cellulose, was admixed with the purified wood cellulose. The resulting admixture was triturated to ensure complete and uniform wetting, and then spread thinly in glass drying trays. The admixture was dried under vacuum (0.5 mm.) at a temperature of 30 centigrade. Upon conclusion of the drying step, the penicillin impregnated wood cellulose was passed through a number 60 size mesh screen and added to the equivalent weight of the dried, screened ethyl cellulose. The resulting admixture was mixed thoroughly and assayed for potency. (Where necessary, the potency is now adjusted by the addition of a calculated amount of a 50-50 mixture of dried, screened, purified wood cellulose and ethyl-cellulose.) The adjusted admixture was mixed thoroughly and then compressed into inch tablets, each weighing mg. The upper and lower tablet punches were embossed with a p I so that the designation was impressed on both sides of each tablet disc as it was formed. The bulk tablets were dried under vacuum (0.5 mm.) at a temperature of degrees centigrade to a final moisture content of 0.6 to 0.8%.

Each of the resulting tablets contained 10 units of penicillin, the entire batch producing 4,000 tablets. Assaying by established procedures (see Grove and Randall) establishcd a variation of less than five percent (5%) in the penicillin potency of the individual tablet discs within the entire lot. Uniformity was further confirmed by a standard performance test. This consisted of comparing the zone sizes produced by fifty tablet discs (randomly selected from the batch) on plates prepared as for the penicillin assay as described by Grove and Randall.

The preparation of a concentrated pre-mix of the antibiotic or other chemical agent on the carried is advantageous in that sufficient concentrate can be conveniently prepared for production of several batches of tablet discs and/or for the manufacture of two or more different strengths. merely by the process of proper dilution.

Especially in larger production batches, e.g. 25,000- 100.000 tablets, it is preferable to employ this procedure, i.e., to admix the solution of antibiotic with about onethird of the final quantity of carrier, e.g. purified wood cellulose, in the proportion of l gm. of carrier to 1 ml. of antibiotic solution. Subsequent dilution and utilization of this concentrate is shown in the following example:

Example II.Tclmcyc/ine Tablet Disc (5 meg. Each) The following components were utilized:

'letracycline (in anhydrous reagent grade methanol containin i750 micrograms (40% overagc) of tetracycline per milliliter)..milliliters 200.00

The tetracycline was dissolved in the anhydrousmeth anol using clean volumetric glassware. The tetracycline solution (200.0 milliliters) was admixed with 200 grams of the purified wood cellulose. The resulting admixture was triturated to ensure complete and uniform wetting, and then spread thinly in glass drying trays. The admixture was dried under vacuum (0.5 mm.) at a temperature of 30 degrees Centigrade. Upon conclusion of the drying step, the tetracycline impregnated wood cellulose was passed through a number 60 size mesh screen and added to the balance (425 grams) of the purified wood cellulose and 625 grams of the dried, screened ethyl cellulose. The resulting admixture was mixed thoroughly and assayed for potency. The potency was adjusted by the addition of a calculated amount of a 50 mixture of dried, screened, purified wood cellulose and ethyl cellulose. The adjusted admixture was mixed thoroughly and then compressed into one-quarter inch diameter tablets, each Weighing 25 milligrams. The upper and lower tablet punches were embossed with a T so that this designation was impressed on both sides of each tablet disc as it was formed. The bulk tablets were dried under vacuum (0.5 mm.) at a temperature of 30 degrees centigrade to a final moisture content of 0.6 to 0.8%.

Each of the resulting tablets contained 5 micrograms of tetracycline. the entire batch producing 50,000 tablets (theoretical yield). Assaying by established procedures (Grove and Randall) established a variation of less than live percent (5%) in the tetracycline potency of the individual tablet discs within the entire lot. Uniformity was further confirmed by a standard performance test. This consisted of comparing the zone sizes produced by fifty tablet discs, randomly selected from the batch, on plates prepared as for the tetracycline assay as described by Grove and Randall.

In using the novel test devices of the present invention, the particular organism to be tested is first streaked on freshly prepared culture plates. Petri plates, mm. in diameter, are preferably utilized, with approximately 20 ml. of culture medium per plate being used. Included among the wide variety of culture media that are suitable are the Oxoid Sensitivity Test Agar, Difco Blood Agar Base, and a Tryptose-thiamine agar. Preferably, about 0.1 ml. of a sixteen to twenty-four hour broth culture of the selected organism is used for streaking.

Next, the tablet discs, each containing a different chemical agent, for example a series of antibiotic discs each containing chloramphenicol, erythromycin, tetracycline, penicillin, and streptomycin, respectively, are placed on the inoculated plates approximately 25 mm. apart. Within ten to fifteen seconds, the tablet discs asa result of rapid absorption of moisture adhere firmly to the surface of the medium. The plates are then inverted, and incubated for l618 hours time at 32-37 centigrade.

Upon conclusion of the incubation period, the results are interpreted, based upon the zone of inhibition appearing around each of the individual tablet discs. It is imperative that the tablet disc not dissolved or otherwise fracture or disintegrate so that the identity of the particular chemical agent stamped or impressed directly on the tablet disc remains legible during the entire test period. An organism is characterized as being sensitive to the particular chemical agent if a characteristic clear zone of inhibition appears around both the high and low potency tablet discs. An organism may be considered resistant to the chemical agent in a particular tablet disc if no zone appears around either the high or the low potency tablet disc, respectively. Where a characteristic clear zone around the high potency but essentially no zone around the low potency tablet disc is observed, the organism is considered as being moderately resistant to that particular chemical agent.

In the prepartion of the tablet discs, the carrier. component is preferably a highly purified wood cellulose (such as the Solka-Flocs). The carrier functions as a medium for rapidly absorbing moisture from the culture medium inoculated with the micro-organism to be tested. This permits ready diffusion of the chemical agent directly into the medium. In addition, the carrier functions in the preferred method of preparing tablet discs as 2. vehicle in which the antibacterial or other chemical agent is pre-mixed. Among the various grades of highly purified wood cellulose found operative were the Solka-Floc BW 40, Solka-Floc BW 100 and Solka-Floc BW 200. In some instances, other materials have been found to provide the satisfactory preferential moisture absorption that is required of the carrier component. Such materials as polymerized organic salts of sulfonic acids of the alkylaryl type (Darvan #1), dextrin polymer (Frodex 15), finely ground calcium sulfate (English Terra Alba) and pyrophyllite (Pyrax) have been found operable.

In general, a tablet disc prepared from only the admixture of the chemical agent with the selected carrier does not produce satisfactory results. For example, a tablet disc made up entirely of purified wood cellulose (Solka- Floc BW 200) and the chemical agent swells readily and cracks apart, thus losing its identity during the incubation y period following implanting in the culture medium containing the micro-organism. Of all the materials examined, only the finely ground calcium sulfate (English Terra Alba) showed promise as a carrier that might eliminate the need of the second, binder component.

The binder component of the novel test device functions to control overall moisture or liquid absorption by the tablet disc while it is in contact with the culture medium. In addition, it functions as a hardener and lubricant in the actual manufacture of the tablet disc. Preferably, certain chemically modified cellulose derivatives are employed, especially the ethylcelluloses. Other binders found operative include sodium silieo aluminate (Zeolex Number 7), colloidal silicon oxide (Pharmasorb), cellulose acetate phthalate and celluloseaeetate hydrogen phthalate. Certain silicone resins and solid polyethylene glycols that are not microbiologically active have also been found to be satisfactory. The employment of the binder component in approximately an equivalent weight to the carrier results in the formation of a reasonably hard, cohesive tablet disc that readily withstands normal handling.

Attempts to utilize the ethylcelluloses and other chemically modified cellulose derivatives and their operative substitutes with only the chemical agent, while excluding the presence of the carrier. proved unsuccessful. It has been found that many of the chemical agents in their appropriate solvent solutions cannot be readily admixed with the binder materials. In some instances, a gummy mass resulted while in others the antibiotic or other chemical agent remained unabsorbed on the binder.

In addition to being inert to the micro-organism, organism or like thing whose sensitivity is being investigated, the binder and carrier are each preferably chemically inert, i.e. they each will not interact chemically with the chemical agent being tested. In general, it is important that the binder and carrier not interfere adversely with either the chemical agent or the micro-organism.

While ratios by weight of carrier to binder of 50 to 50 are preferred. it has been found that ratios of between 40 to 60 and 60 to 40 of carrier to hinder produce satis factory results. In some circumstances, tablet discs have been developed in which as little as ten percent (10%) hinder or carrier. respectively, has been used.

It is to be understood that the invention is not to be limited to the exact devices, compositions and methods shown and described as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the scope of the appended claims.

We claim:

I. A test device for use in the determination of the sensitivity of microorganisms to a chemical agent which comprises:

(a) a line mesh, microbiologically inert, purified celluiose carrier material;

(b) a chemical agent uniformly absorbed in the carrier material; and

(u) a microbiologically inert chemically modified ceilulose derivative binder in admixture with the carrie material having absorbed therein the chemical agen the test device being an integral composition of matter in thin tablet form, being non-disintegratable by absorbed moisture and with a moisture content of less than about one percent, the weight ratio of carrier material to binder material being between about 10 to 90 and 90 to 10, and the chemical agent, binder material and carrier material being uniformly dispersed in the test device.

2. Claim 1 wherein the binder material is ethyl cellulose.

3. Claim 2 wherein the ratio of carrier material to binder material is between about 40 to 60 and 60 to 40 weight percent.

4. Claim 3 wherein the carrier material is about 60 mesh.

5. A test device for use in the determination of the senr sitivity of micro-organisms to a chemical agent which comprises:

(a) a fine mesh, moisture absorbing, microbiologically inert carrier material; (h) a chemical agent uniformly absorbed in the carrier material; and (c) a mierobiologically inert binder material in admixture with the carrier material having absorbed therein the chemical agent, the carrier material being preferentially wettable by water in relation to the binder material, the test device being an integral composition of matter in thin tablet form, being non disintegratable by absorbed moisture and with a moisture content of less than about one percent, the weight ratio of carrier material to binder material being between about. 10 to 90 and 90 to l0 and the chemical agent. binder material and carrier material being uniformly dispersed in the test device. 5. Claim 5 wherein the carrier material. is purified wood cellulose.

7. Claim 5 wherein the binder material is ethyl cellulose.

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Kwan et at: Factors Atlect n" Tablet Disintegration." April 1957. J. American Phi-.1 .utical Association, vol. 46, New York. page 239. 

1. A TEST DEVICE FOR USE IN THE DETERMINATION OF THE SENSITIVITY OF MICRO-ORGANISIMS TO A CHEMICAL AGENT WHICH COMPRISES: (A) A FINE MESH, MICROBIOLOGICALLY INERT, PURIFIED CELLULOSE CARRIER MATERIAL; (B) A CHEMICAL AGENT UNIFORMLY ABSORBED IN THE CARRIER MATERIAL; AND (C) A MICROBIOLOGICALLY INERT CHEMICALLY MODIFIED CELLULOSE DERIVATIVE BINDER IN ADMIXTURE WITH THE CARRIER MATERIAL HAVING ABSORBED THEREIN THE CHEMICAL AGENT, THE TEST DEVICE BEING AN INTEGRAL COMPOSITION OF MAT TER IN THIN TABLET FORM, BEING NON-DISTEGRATABLE BY ABSORBED MOISTURE AND WITH A MOISTURE CONTENT OF LESS THAT ABOUT ONE PERCENT, THE WEIGHT RATIO OF CARRIER MATERIAL TO BINDER MATERIAL BEING BETWEEN ABOUT 10 TO 90 AND 90 TO 10, AND THE CHEMICAL AGENT, BINDER MATERIAL AND CARRIER MATERIAL BEING UNIFORMLY DISPERSED IN THE TEST DEVICE. 